Treg cell diversity and homeostatic control

Treg 细胞多样性和稳态控制

基本信息

批准号：
10551213
负责人：
CHRISTOPHE O. BENOIST
金额：
$ 51.25万
依托单位：
HARVARD MEDICAL SCHOOL
依托单位国家：
美国
项目类别：
财政年份：
2020
资助国家：
美国
起止时间：
2020-02-11 至 2025-01-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10551213
关键词：
Ablation Acute Affect Antibodies Arthritis Autoimmune Autoimmune Diseases Autoimmunity Back Bar Codes Biological Markers Blocking Antibodies CD4 Positive T Lymphocytes Cell Count Cell Maintenance Cell Separation Cell Survival Cell physiology Cell surface Cells Cellular Indexing of Transcriptomes and Epitopes by Sequencing Cellular biology Clustered Regularly Interspaced Short Palindromic Repeats Complex Cues Cytokine Receptors DNA Data Development Dissection Equilibrium FOXP3 gene Family Family member Feedback Flow Cytometry Genes Goals Grant Growth Factor Heterogeneity Homeostasis Human IL2 gene IL7 gene Immune Tolerance Immunologic Surveillance Immunooncology Immunotherapy In Vitro Inbred Strains Mice Individual Infiltration Inflammation Inflammatory Joints Knowledge Lesion Locales Location Lymphoid Tissue Maintenance Malignant Neoplasms Mediator Membrane Proteins Mining Mus Organ Pathway interactions Patients Peripheral Phenotype Phosphorylation Play Population Process Proliferating Proteome Regulation Regulatory T-Lymphocyte Rest Role Signal Transduction Sorting Structure Surface T cell differentiation T-Lymphocyte Subsets TSLP gene Testing Therapeutic Thymus Gland Tissues Tumor Necrosis Factor Receptor Variant Work antagonist base biomarker panel combinatorial cytokine immune function immunoregulation in vivo interest loss of function lymphoid organ member morphogens novel overexpression receptor segregation single-cell RNA sequencing stem cells therapeutic target transcription factor transcriptome transcriptomics tumor

项目摘要

CD4+ T regulatory (Treg) that express the transcription factor FoxP3 cells play key and non-redundant roles in maintaining immunologic tolerance and controlling inflammation. Treg proportions within CD4+ T cells are under tight homeostatic control. There is considerable diversity to Treg cells, with differential ability to control certain immune functions, and even tissue-resident Tregs with extra-immunologic functions. Our recent recent single-cell RNAseq work has charted these finely, showing that Treg subsets express different effector molecules. Treg subset structure appears very similar in mouse and humans. We hypothesize that Treg subsets in lymphoid tissues respond to different homeostatic cues, and that these different inputs condition specific Treg functions. (1) Our single-cell transcriptomics data, which highlighted the diversity of Treg subphenotypes in an unbiased fashion, identified some cell surface markers for identification and sorting, but we will expand this base by performing a combined transcriptome/proteome analysis (scRNAseq combined with surface protein quantitation by DNA-tagged antibodies - CITE-seq) to identify a panel of flow cytometry markers that accurately distinguish Treg subphenotypes, in both mouse and human. This will relate this novel landscape to prior knowledge on Treg subsets, provide biomarkers, and enable cell sorting for functional experimentation. We will assess the function of Treg subsets in regulating other immunocytes (proliferation and differentiation), assess their developmental origin and stability (thymic or peripheral, plasticity?), and how they vary in autoimmune or tumor lesions. (2) IL2 is the canonical controller of Treg cellsbut other common- cytokines (IL7, 15, 21 and TSLP) and IL33 also affect Tregs. Our results show a strikingly divergent distribution of cytokine receptors among Treg subsets, suggesting that different growth factors support them. We will use both gain- (in vivo cytokines administration) or loss-of-function (blocking antibodies) strategies to modify Treg subset balance, read out by flow cytometry (STAT phosphorylation, Treg subset number and activation) and scRNAseq. (3) Most members of the large TNF Receptor (TNFR) superfamily are over-expressed in Treg cells, and some have been shown to affect Treg maintenance and survival, but a comprehensive picture is lacking. Our data show differential representation of TNFR across Treg subsets. We hypothesize that TNFR molecules network with cytokine receptors to provide specific trophic signals to distinct Treg subsets. We have developed a powerful CRISPR-based in vivo screen, based on barcoding stem cells, to evaluate how sets of genes partake in Treg homeostasis. We will analyze the role of the entire TNFR family on maintenance of different Treg subsets, at baseline in different lymphoid tissues and after stimulation by trophic cytokines. We will also test several TNFRs combinatorially, to search for redundancy, complementarity or antagonism. These results will provide a clarification and deeper understanding of the diversity of Treg populations, their control by cytokines or surface receptors, and how they might be specifically manipulated for therapeutic intent.

表达转录因子 FoxP3 的 CD4+ T 调节 (Treg) 细胞发挥关键且非冗余的作用维持免疫耐受和控制 CD4+ T 细胞内 Treg 比例的作用。 Treg 细胞处于严格的稳态控制之下，具有不同的能力。控制某些免疫功能，甚至具有额外免疫功能的组织驻留性Treg。最近的单细胞 RNAseq 工作详细地描绘了这些，表明 Treg 亚群表达不同的效应子 Treg 分子的子集结构在小鼠和人类中显得非常相似。淋巴组织中的亚群对不同的稳态信号做出反应，并且这些不同的输入条件 (1)我们的单细胞转录组数据，突出了Treg的多样性。以公正的方式分析亚表型，鉴定了一些细胞表面标记以进行识别和分类，但是我们将通过执行转录组/蛋白质组分析（scRNAseq 组合通过 DNA 标记抗体进行表面蛋白定量 - CITE-seq）来鉴定一组流式细胞仪准确区分小鼠和人类 Treg 亚表型的标记物这将与这篇小说相关。景观 Treg 子集的先验知识，提供生物标志物，并实现功能性细胞分选我们将评估 Treg 亚群在调节其他免疫细胞（增殖和免疫细胞）中的功能。分化），评估它们的发育起源和稳定性（胸腺或外周，可塑性？），以及它们如何 (2) IL2 是 Treg 细胞的典型控制器但其他常见 - 细胞因子（IL7、15、21 和 TSLP）和 IL33 也影响 Tregs 我们的结果显示出明显不同的分布。 Treg 亚群中细胞因子受体的数量，表明我们将使用不同的生长因子支持它们。修饰 Treg 的获得（体内细胞因子施用）或功能丧失（阻断抗体）策略子集平衡，通过流式细胞仪读出（STAT 磷酸化、Treg 子集数量和激活）以及 scRNAseq. (3) 大 TNF 受体 (TNFR) 超家族的大多数成员在 Treg 细胞中过度表达，有些已被证明会影响 Treg 的维持和存活，但缺乏全面的了解。我们的数据显示 TNFR 在 Treg 亚群中的差异表达，我们认为 TNFR 分子具有差异性。我们开发了与细胞因子受体的网络，为不同的 Treg 亚群提供特定的营养信号。强大的基于 CRISPR 的体内筛选，以条形码干细胞为基础，评估基因组如何我们将分析整个 TNFR 家族在维持不同的 Treg 稳态中的作用。我们还将在不同淋巴组织的基线和营养细胞因子刺激后观察 Treg 亚群。组合测试多个 TNFR，以寻找这些结果的冗余性、互补性或拮抗性。将澄清和更深入地了解 Treg 群体的多样性及其控制细胞因子或表面受体，以及如何专门操纵它们以达到治疗目的。